PROJECT SUMMARY Hematopoiesis is the process of blood production that is continuously active in humans for their entire lifespan. Mature effector cells are continually being exhausted leading to a requirement for a massive daily production of leukocytes, erythrocytes, and platelets. This blood production occurs primarily in the bone marrow through a cellular differentiation hierarchy initiated and maintained by self-renewing hematopoietic stem cells (HSCs) that give rise to a variety of progenitor cells and eventually all the mature, terminally differentiated cells of the blood. HSCs are able to produce blood cell progeny for the entire life of an individual, undergoing self-renewing cell divisions that maintain HSC numbers. These two features, self-renewal and multipotent differentiation, represent the key characteristics of HSCs. HSC self-renewal in particular has been of great interest for both biological mechanistic studies and potential translational applications. In clinical practice, HSCs are the fundamental unit of hematopoietic cell transplantation (HCT) utilized in the treatment of both benign and malignant blood disorders. Although not yet utilized in clinical practice, genome editing approaches to the treatment of blood disorders will require ex vivo expansion of edited HSCs prior to transplantation, highlighting the critical need to understand HSC self-renewal. For biological studies, the mouse has been used as the primary experimental model for the investigation of HSC self-renewal. A number of molecular pathways have been implicated in this process including Wnt/beta-catenin, Notch, and Bmi1, and our group and others demonstrated that cohesin-deficiency results in increased HSC self-renewal. Recently, a number of studies with both mouse and human cells have implicated an unusual homeobox family member, Hopx, as a potential regulator of HSC self-renewal. HSCs predominantly reside in the bone marrow where they are contained in a complex microenvironment consisting of cellular components including osteoblasts, mesenchymal stromal cells (MSCs), endothelial cells, and others, as well as growth factors, cytokines, adhesion molecules, and extracellular matrix. The nature of the hematopoietic microenvironment and specifically the mouse HSC niche has been an area of intense investigation, but much less is known about the human HSC niche. Overall, our current understanding of human HSC self-renewal is much less extensive than in the mouse. Here, we propose to identify and investigate novel regulatory determinants of human HSC self-renewal through several hypotheses and approaches. First, we will investigate epigenetic DNA regulatory elements that are critical for human HSC self-renewal by investigating chromatin accessibility in cohesin-deficient HSCs. Second, we will investigate the role and mechanisms of action of Hopx as a regulator of human HSC self-renewal, including possible links to the cell cycle and Wnt pathway. Finally, we will use a novel humanized ossicle xenograft model in conjunction with lineage tracing to visualize bone marrow niches associated with human HSCs. Together, these aims should greatly expand our understanding of human HSC self-renewal.